The present invention relates to a method allowing the identification of signatures corresponding to a specific sequence of double-stranded nucleic acid.
In the field of DNA sequence analysis, the present invention relates to a method which makes it possible to accelerate or bypass the process of genomic DNA sequencing, a problem which is of considerable industrial interest. The method according to the present invention allows the identification of specific signatures of a sequence on a long DNA fragment. This method makes it possible in particular to rapidly search for the presence of particular structures in the base sequence and/or the quality of the pairings which may be partial and, in particular, have mismatches. This method also allows analysis and comparison of the genomic differences among various patients.
It is understood that the invention also applies to single-stranded DNA-single-stranded DNA duplexes, perfectly paired or not perfectly paired, or alternatively to single-stranded DNA-single-stranded RNA duplexes, perfectly paired or not perfectly paired, or alternatively to single-stranded RNA-single-stranded RNA duplexes, perfectly paired or not perfectly paired. Furthermore, the duplex may consist of the at least partial re-pairing of two single strands obtained from samples of different origins. Finally, the invention also applies to the secondary structures of a sole single-stranded DNA or of a sole single-stranded RNA.
To clarify the disclosure, there is used in the text which follows the specific example of a double-stranded DNA, it being understood that the above generalizations can be easily achieved by simply developing the methods described.
A xe2x80x9csignaturexe2x80x9d makes it possible to xe2x80x9ccharacterizexe2x80x9d a DNA sequence.
By way of example, the pairing of the G and C bases involves three hydrogen bonds, whereas the pairing of the T and A bases involves only two bonds; under these conditions, if the DNA double helix is mechanically opened by separately pulling the ends of two strands on the same side of an isolated DNA molecule, it can be expected that the signal corresponding to the forces exerted depends on the sequence.
Thus, the implementation of the present invention has made it possible to demonstrate that the separation of two paired strands essentially results in the obtaining of a xe2x80x9csignaturexe2x80x9d, that is to say a set of specific and local information which is linked to the sequence and/or to the state of pairing.
Unexpectedly, during the opening of a duplex, the signal in force may exhibit in particular serrated structures, with a gentle rise and a more abrupt descent, where the opening occurs, in part, in short bursts. The forces measured during the opening therefore exhibit a set of particular characteristics, or signature, a complex combination of the sequence where the regions rich in GC are harder to open than the regions rich in AT, and of the mechanical stiffness of the system (molecular construction and system of measurement), which come into play to induce events where several contiguous bases open more rapidly.
By modifying the mechanical stiffness of the system, it is possible to increase (at low stiffness), or decrease (at high stiffness) the effects of instability and to obtain a set of signatures for the same sequence.
Thus, a xe2x80x9csignaturexe2x80x9d does not constitute a base-to-base sequencing of an entire sequence, but makes it possible to obtain a set of specific and local information, linked to the sequence and/or to the state of pairing.
Measurements of force on isolated molecules of DNA currently constitute a very active field (see references 1 to 11). For a range of forces extending from one subpicoNewton to tens of picoNewtons, which forces are typically involved in weak molecular interactions, sensitive devices for the measurement of forces such as optical tweezers (Svoboda et al., 1993; Yin et al., 1996) or flexible microneedles are being increasingly used.
In a typical configuration intended to open DNA, the molecules may be specifically anchored on two solid substrates (microscope slide, micropipette, microparticle). One of the ends being attached and the other being connected, for example via a particle, to a device for measuring force.
Patent WO 94/23065 describes a device for measuring force, base-to-base, during mechanical opening of DNA using an atomic force microscope.
However, the base-to-base resolution is fraught with basic limitations linked to thermal noise (Thompson al., 1995; Viovy et al., 1994).
Moreover, carrying out the opening in practice exhibits operational difficulties, in particular:
the design of the molecular construction,
the chemistry of the surfaces and their preparation,
the manner of selecting the place where the measurement is carried out.
In particular, it is crucial, in order to carry out these experiments, to reduce:
the level of adsorption of the study molecule on the surfaces (microscope slide or particle);
the adhesive interactions between the particles and between the particles and the surfaces.
Finally, since a single measurement can take several tens of minutes, it is necessary to have a very efficient selection mechanism in order to macroscopically identify the most useful points so as to carry out the measurements despite the fact that the chemistry of the surfaces is imperfect, and of course, despite the fact that the constructions themselves may be imperfect.
The present invention includes the aspects relating to the chemistry of the surfaces and the specific constructions so as to obtain satisfactory results.
More particularly, the present invention relates to a method for characterizing a nucleic acid duplex comprising two at least partially paired nucleic acid sequences, characterized in that at least one xe2x80x9csignaturexe2x80x9d of said duplexes, which is linked to the variation in force necessary to unpair, respectively re-pair, said two sequences is recorded, and in that the signature obtained or some characteristics thereof is compared with references.
It is understood, by way of example, that a reference may consist of another measurement, a combination of several measurements, or alternatively of a numerical calculation, for example such as that which will be described in the text which follows, or alternatively a combination of the preceding cases.
xe2x80x9cNucleic acid duplexxe2x80x9d is understood to designate any type of DNA/RNA duplex as mentioned above.
More particularly, the present invention relates to a method characterized in that the 5xe2x80x2 end of one of the strands of the duplex is attached to a support 1 and the 3xe2x80x2 end of the other strand of the duplex is attached to a support 2, the variation in force necessary to unpair, respectively pair, said sequences being measured by moving apart, respectively bringing closer, said supports 1 and 2, characterized in that, during the attachment, the point of attachment of the 3xe2x80x2 end on the support 2 and the point of attachment of the 5xe2x80x2 end on the support 1 are linked together by a molecular string having a length of at least 0.03 xcexcm, and preferably of between 0.5 and 30 xcexcm.
Within the framework of the present invention, and to clarify the notations, the 5xe2x80x2 end of one of the strands of the duplex will be called xe2x80x9c5xe2x80x2 end of the duplexxe2x80x9d and the 3xe2x80x2 end of the other strand of the duplex will be called xe2x80x9c3xe2x80x2 end of the duplexxe2x80x9d, and, in this definition, the strands can of course be reversed.
xe2x80x9cMolecular stringxe2x80x9d is understood to designate both the distance calculated on the molecule (it being possible for the latter to be folded), for example the spacer arm (mode I), and the spatial distance (measured in particular along the strands between the two points of attachment (mode II).
Supports 1 and 2 may also be interchanged in the developments which follow.
Advantageously, the method in accordance with the invention is characterized in that the structure of the duplex is locally modified in particular by the creation of a triplet, a complex or the attachment of a protein.
A few terms which will later be used in accordance with their respective definition are defined below.
Given that the experiments are carried out in a buffer, a xe2x80x9cwellxe2x80x9d is a component intended to contain a small volume of buffer and it may advantageously incorporate or contain one or more components, which are detachable or otherwise, called xe2x80x9cslidesxe2x80x9d.
The xe2x80x9cslidexe2x80x9d is a solid component which is flexible or rigid in nature and has the following characteristic features:
a) At least part of this slide is in contact with an essentially aqueous buffer, and at least a fraction of this part in contact is functionalizable on a surface of at least 0.01 xcexcm2, and preferably between 1 xcexcm2 and 10 cm2.
b) The slide being in place in the well, it is possible to bring closer, in order to obtain an image of the functionalizable part in contact with the buffer, either an immersion microscope lens in aqueous media, or an immersion microscope lens in oil and, in this case, the slide may be advantageously thin, flat and substantially transparent.
By way of specific example, the slide may be a microscope coverslip, a polymer coverslip or a particle maintained by a micropipette.
A well may be advantageously designed so as to be able to receive a xe2x80x9cmicropipettexe2x80x9d.
A xe2x80x9cmicropipettexe2x80x9d is a solid component, flexible or rigid in nature, of which one end may be attached to a fixed or moving system, and of which at least the other end is intended to be immersed in a liquid. It is substantially elongated in appearance and its characteristic diameter is greater than 0.01 xcexcm, preferably between 0.1 xcexcm and 2 mm, and it may be advantageously hollow.
The supports envisaged may be either slides, particles, micropipettes or a component of an apparatus for measuring force.
In the method according to the present invention, the manipulations are preferably carried out with specific molecular constructs, advantageously attached, between a slide for example, and microparticles, or alternatively by attachment of the ends to two particles.
This method essentially comprises two embodiments.
More particularly, in the first embodiment (mode I) of the method, it is a method in which the 3xe2x80x2 end of the duplex to be characterized (respectively the 5xe2x80x2 end) is attached to one of the supports via a spacer arm having a length of at least 0.03 xcexcm, preferably of between 0.5 and 30 xcexcm.
In this method, one of the characteristics is that the DNA duplexes intended to be opened are not directly attached to a support, but are attached via at least one spacer arm placed either on the support 1, or on the support 2, or on both. Indeed, the use of a spacer arm makes it possible to select the site and/or the time for carrying out the measurement. It is possible, for example, to adapt to this type of molecular construct the selection and measurement technique used by Smith et al. (1996). It is also possible to carry out multiple anchorages on a slide.
Under these conditions, the particles linked to the slide via a molecular construct have some freedom of movement. Of course the length of the spacer arm may be advantageously adjusted in relation to the spatial resolution of the system for locating the position of the beads.
When a weak force is simply applied to the particles, the DNA will not open and it will be possible to observe the following characteristic movement:
A particle moves but is then blocked in its movement by the presence of the spacer arm. The characteristics of this movement make it possible to select the systems of interest on which a measurement cycle can be applied and make it possible in particular (i) to eliminate the imperfect attachments; in particular if the particle starts to adsorb to the slide, or if the DNA starts to adsorb, the characteristics of the movements will be more limited, and therefore different; (ii) during the slightly abrupt phase of sticking of a measuring microneedle, the relative freedom of movement of the particle makes it possible to avoid the pulling out of the point of attachment.
The spacer arm may consist of double-stranded DNA, of 100 to 500 bases, that is to say about 0.03 xcexcm to 0.2 xcexcm, but preferably the spacer arm will correspond to 1 kb or will even comprise from 5 to 100 kb, which will approximately correspond to a clearance of 1.5 to 30 xcexcm, but it is also possible to envisage spacer arms which would not consist of a double-stranded DNA, but which would consist of other components of the synthetic polymer type, of the protein type or alternatively of other, for example polysaccharide, polymers.
Thus, just before the measurement, the sample which is under the microscope consists of:
nonattached mobile particles;
particles which are immobile on the slide;
particles with a limited movement.
Of course, only the latter particles are of interest. In order to improve the selection of these particles, it is possible to apply to them either a magnetic field gradient, in particular a magnet, if particles have been chosen on which a magnetic gradient exerts a force, or a fluid-type field, or an electric field, or an electric field gradient. And the particles which have a zone of movement which corresponds to the expected extension of the spacer arm in the absence of undesirable attachments of the spacer arm or of the DNA sequence with the surface or with the particles themselves, are chosen at this time.
In general, the length of the spacer arm, which makes it possible to locate the particles which may be the subject of a study, is known. Techniques intended to allow grafting on the ends of molecular constructs are known; they are essentially systems using a ligand/receptor combination, or alternatively covalent bonds. Preferably, the ends to be grafted are biotinylated or functionalized with a ligand such as dig, whereas the glass slides for example, or the particles are respectively coated with avidin or streptavidin or an antidig antibody.
In general, to attach the end of the nucleic acid sequence to the support, it is possible to use:
a covalent interaction
an antigen/antibody interaction
a ligand/receptor interaction
an avidin or streptavidin/biotin interaction.
The optional attachment of the particle or of the support to the device for measuring force may be carried out by any means, in particular by bonding, as above, but also by sticking or by magnetic influence using appropriate beads.
More specific constructs will be described in the examples below.
The spacer arm may of course be placed either on the first support, a slide consisting of a glass surface for example, but it is possible to envisage an attachment to the particle via a spacer arm; in this case, the method is characterized in that the 5xe2x80x2 end of the duplex to be characterized, respectively the 3xe2x80x2 end, is attached to the support 2 via a spacer arm having a length of at least 0.03 xcexcm, but preferably of between 0.5 xcexcm and 30 xcexcm; in this case, one of the ends of the duplex may be directly attached to the slide. In order to further improve the method, it is possible to envisage the use of two spacer arms, one in relation to the surface or support 1, the other in relation to the particle or support 2.
In the second embodiment (mode II), the support 2 is attached to the 3xe2x80x2, respectively 5xe2x80x2, end, while the duplex to be characterized has been at least partially denatured and the ends of the two unpaired nucleic acid sequences comprising it have been partially spatially moved apart by a distance of at least 0.03 xcexcm, preferably of between 0.5 and 30 xcexcm.
Preferably, the free ends of the duplex are linked by a hairpin-shaped sequence which makes it possible to denature the entire duplex.
More specifically,
the duplex being blocked by a hairpin, each of the free ends is functionalized, for example, with biotin and dig,
then a particle comprising, for example, streptavidin is attached to the corresponding end,
this bead is then immobilized by any, for example mechanical (micropipette), means,
the complex is then denatured and the whole is placed in a stream of fluid,
the end not attached to the bead moves away and an anti-dig treated bead is for example attached to this end; this bead can be approached by any means, in particular optical tweezers.
In the preferred embodiment mode I, the spacer arm will consist of a double-stranded DNA, for example a double-stranded DNA obtained from the xcex phage.
The DNA duplex (DNA 1) for which it is desired to obtain a signature according to the present invention should be connected to the spacer arm. Preferably, the duplex is digested with at least one restriction enzyme, which makes it possible to know the nature of its ends, blunt or cohesive, and in this case the cohesive sequence.
It is then easy for persons skilled in the art to attach the segment to the spacer arm (lambda DNA and the like), for example by a cassette-type system.
An exemplary embodiment is given in FIG. 2B which is a spacer arm cassette system, DNA 2 is a double-stranded DNA of xcex phage completed by:
(i) dig functionalization on one side: this is carried out, for example, with the aid of oligo 3, which is a Cos sequence dig functionalized in 3xe2x80x2 and is covalently attached by hybridization and then ligation;
(ii) the system of adaptors oligo 1 and oligo 2: oligo 1 has a Cos sequence and is attached by hybridization and ligation to DNA 2; oligo 2 is 3xe2x80x2 functionalized with biotin; it has a sequence which is complementary to part of oligo 1 and can therefore attach to it by simple hybridization, the free end of the oligo 1-oligo 2 duplex is chosen so that it is cohesive with the cleavage of the DNA-1 DNA with a restriction enzyme.
By hybridization and then ligation, a covalent bonding of the two 3xe2x80x2 and 5xe2x80x2 strands of one end of DNA 1 is obtained: the 3xe2x80x2 strand to oligo 2 and the 5xe2x80x2 strand to oligo 1.
In the final construct, the dig end is attached to a support 1 and biotin to a support 2. The support 1 may be, for example, a glass coverslip coated with antidig, and the support 2 may, for example, be a microbead coated with streptavidin.
The opening of DNA 1 may be obtained by moving the support relative to the bead, which draws its 3xe2x80x2 strand linked to the bead, and its 5xe2x80x2 strand linked to the coverslip by the spacer arm DNA 2. In this example, it is naturally possible to interchange the positions of the dig and biotin functionalizations.
A cassette assembly as has just been described by way of example may be a component of a diagnostic kit, so as to obtain the construct which allows opening by a simple hybridization-ligation operation.
By way of example, such a technique, applied to the opening of the lambda phage DNA (DNA 1, FIG. 2), with a spacer arm consisting of the double-stranded DNA of a lambda phage (DNA 2, FIG. 2), is described.
In the final construct, the oligo 2 connector may have a free nonpaired region, close to biotin, so as to facilitate subsequent reaction with the microparticles. The entire construct is linked by ligation steps after the various hybridization steps, leaving the possibility of opening the molecule at the biotinilated end. Naturally, other combinations of ligands and of receptors may be chosen, or alternatively multiple ligands of the same type may be used in place of a single one (Cluzel et al., 1996; Strick et al., 1996). Finally, the spacer arm may be naturally placed either between support 1 and DNA to be opened (example given here) or between DNA to be opened and support 2, or a combination of preceding cases. An additional feature of the method is introduced as regards the other end of the molecule to be opened. It may be capped with a cohesive hairpin-shaped oligonucleotide (hairpin-oligo) which avoids the two strands separating when the end of the opening process is reached. This makes it possible to repeat complete opening-closing cycles. It is also possible not to use this additional component and to carry out a partial opening while avoiding completely unpairing the duplex, which advantageously makes it possible to repeat partial opening-closing cycles.
Signature Modification
As will be seen in the text which follows, the signature depends on the total stiffness of the system, that is to say of the measuring system and also of the molecular construct, in particular that of the single strands of the opened molecule. This stiffness parameter can be advantageously varied in order to obtain different signatures for the same sequence.
1) Stiffness of the Measuring System
As regards the lever, it is easy to change it for levers of different stiffness. In the case of a measurement by an optical tweezer, this may be obtained, for example, by simple modification of the intensity or of the position of the trapping laser. Naturally, other modes of operation, in particular with retroaction (for example Finer et al, 1994; Svoboda et al., 1993; Yin et al., 1996), may be advantageously used.
Two examples are given:
(i) In one of these cases, the position of the tweezers may be automatically adjusted by retroaction to prevent the movements of the bead. The variation in the force during the opening results in a variation in the position of the trap.
1 ii) In the other example, the intensity of the tweezers may be controlled by retroaction so as to keep the position of the bead fixed. The variation in the force during the opening results in a variation in the intensity of the laser.
In conclusion, the present invention includes several modes of measurements, potentially resulting in different types of signature. It is therefore possible, for the same sequence, to accumulate several different signatures whose information may overlap and be complementary.
2) Stiffness of the Single Strands
In the same spirit, the present invention includes the modification of the stiffness of the single strands (which are in series in the measurement) by introducing, in the measuring buffer, particular molecules known to modify the stiffness of the single strands.
In particular, there are numerous proteins which have an affinity for the single strands, such as recA or SSBP (Single Strand Binding Protein), and which can increase the stiffness of the single strands. Likewise, oligonucleotides (for example a random sequence population) may also be used.
Other Experimental Conditions Which can be Envisaged
It may be useful to vary the experimental conditions in order to obtain different signatures on the same molecule. Thus, it is possible to vary the temperature and/or modify the buffer used. In particular, some specific buffers may be advantageously used in order to have an action on the stability of the pairings between bases (for example, Rees, 1993).
Modification of the Duplex or of the Single Strands
It is possible to induce particular marks, or imprints, at certain points of the signature. These are obtained by opening a molecule modified by addition of reagent. The term xe2x80x9creagentxe2x80x9d is understood here to mean any molecule capable of establishing a specific interaction with part of the duplex, whether it is open, closed or both open over some parts and closed over others. The modification of the duplex or of the single strands is advantageously chosen so as to locally or substantially modify the force for separating the strands, which may even extend to complete blocking of the opening.
Without as a result being limited thereto, a few examples are given below with:
A)
the introduction of a stronger bond into one or more specific zones of the sequence; this leads, in the signature, to one or more characteristic rises, when the mean opening point reaches these zones;
the introduction of one or more proteins characterized in that they are capable of specifically binding to the duplex, for example to some sequences (such as a methylation enzyme) or alternatively to some structures (such as the binding to a mismatch, with the Mut protein, for example); such a binding can also be used to position a particular reagent (attached to a protein) making it possible to induce an inter-strand xe2x80x9ccross linkxe2x80x9d with a photoactivable group for example;
the introduction of a strengthened inter-strand bond, for example by formation of a triple helix (with a sequence of oligonucleotides or of their analogs); such a bond may also be used to position a particular reagent (attached to the oligo) making it possible to induce an inter-strand xe2x80x9ccross linkxe2x80x9d, with a photoactivable group for example;
the buffer used and/or the choice of the temperature can also substantially modify the force necessary to unpair, respectively re-pair, the duplex.
B)
additional molecules can be caused to interact with the single strands of the partially open nucleic acid molecule, characterized in that they preferably bind to at least one specific site on a single strand; during the reclosing and/or reopening, the signal of this signature is then modified.
Uses of the Signatures
One or more signatures, with imprint or otherwise, corresponding to a given duplex, may be compared with references, consisting either of one or more measurements on another duplex, or with numerical calculations, or a combination of both.
It is thus possible to detect and locate the absence or the presence of one or more of the following parameters given by way of example:
homology
translocation,
deletion,
amplification,
homologous repeat regions,
zone of partial pairing,
specific unit of the sequence,
difference,
mutation,
contigation,
a particular region, identifiable by its GC content
a CpG island.
This method constitutes a sequencing aid. It also allows the analysis and the comparison of the genomic differences among various patients or various DNA samples to be tested.
The invention finally relates to a diagnostic kit intended for carrying out the claimed method and which is described below.
The diagnostic kit is characterized in that it comprises one or more of the following components:
at least one spacer arm,
at least one spacer arm cassette molecular construct,
at least one joining oligonucleotide,
at least one hairpin-shaped oligonucleotide,
at least one restriction enzyme,
at least one molecular construct intended to attach the strands of the duplex to be opened to components of the apparatus for measuring force,
a support which is attachable to the functionalized end of the spacer arm,
at least one well,
at least one slide,
at least one type of functionalized and advantageously magnetizable beads intended to be attached to a functionalized component of the duplex to be opened,
at least one magnet,
at least one buffer,
at least one ligation enzyme,
at least one ligation buffer,
at least one column for separation by centrifugation.